Excessive alcohol consumption is the third leading cause of preventable death in the US. To date, studies that examine the molecular mechanisms of alcoholic liver disease are performed using models in which the genetic and environmental backgrounds are unaffected by prevalent pre-existing pathologies or underlying risk factors observed in human populations. Importantly, these models do not reflect human populations in two important aspects: 1) widespread hyperlipidemia and 2) pervasive exposure to airborne environmental toxicants like second-hand cigarette smoke, a.k.a. environmental tobacco smoke (ETS). This is important because these risk factors are ubiquitous and epidemiologic studies show that hyperlipidemia and cigarette smoke have additive or synergistic effects to exacerbate alcoholic liver disease. Indeed, the combined effect of chronic alcohol with other co-morbidity risk factors on hepatic physiology and injury remains poorly defined. Accordingly, this application has the goal of investigating the molecular pathways and targets through which ETS on a background of hyperlipidemia accelerates and amplifies alcoholic liver injury. This project builds on new findings in an animal model, which reveals a critical role of alcohol, cigarette smoke, and hyperlipidemia to amplify liver injury via enhanced hypoxic and oxidative/nitrative stress, which all have mitochondrial involvement. Indeed, in the course of these studies a central role for mitochondrial dysfunction has emerged. Findings in support of this are: (1) Mitochondrial DNA damage is highest in mice exposed to alcohol and ETS; (2) Alcohol and ETS decrease cytochrome c oxidase protein and activity; and (3) Alcohol and ETS increase liver iNOS and oxidative protein modifications leading to a loss of function. A recently emerging and novel aspect of mitochondrial function is the role the organelle plays in key redox signaling pathways, which regulate cellular responses to metabolic, hypoxic, and oxidative stress. These findings raise the provocative hypothesis that defects in mitochondrial function disrupt these signaling pathways and contribute to the pathophysiology of alcohol hepatotoxicity and environmental insults like ETS. Based on this, we hypothesize that combined exposure to alcohol, cigarette smoke, and hyperlipidemia increases mitochondrial dysfunction and ROS production triggering activation of HIF1a, which in turn contributes to amplify steatosis. This hypothesis will be tested in three aims: (1) Test the hypothesis that alcohol, cigarette smoke, and hyperlipidemia increase mitochondrial dysfunction and promote the mitochondrial permeability transition; (2) Determine the individual and combined effects of alcohol, cigarette smoke, and hyperlipidemia on mitochondrial ROS/RNS production and modifications to respiratory proteins; and (3) Test the hypothesis that mitochondrial ROS/RNS contribute to HIF1a activation and steatosis from alcohol, cigarette smoke, and hyperlipidemia. These studies will reveal and mechanistically define the additive or synergistic relationship between alcohol and other co-morbidity risks like cigarette smoke and hyperlipidemia in the pathophysiology of alcoholic liver disease. PUBLIC HEALTH RELEVANCE: Alcohol abuse is estimated to be the third leading cause of preventable death in the US. Importantly, new studies report an increasing prevalence and severity of liver diseases from all causes in the US. It has also become clear in recent years that other pre-existing medical conditions like type 2 diabetes and high cholesterol along with exposure to toxicants like cigarette smoke can worsen liver disease in the chronic alcohol consumer. The studies in this application are important as they will attempt to answer the question: What are the roles of other environmental and metabolic factors in worsening liver disease in the chronic alcohol consumer? Research in this area will help medical professionals better understand the causes of liver disease and lead to the discovery of new treatments for patients suffering from liver diseases associated with diabetes, obesity, hepatitis C, and alcoholism.